International Journal of Mycobacteriology 3 ( 2 0 1 4 ) 1 7 8 1 8 3

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Sputum conversion at the end of 8 weeks among

category 1 tuberculosis patients: How reliable
are the peripheral laboratory results?

Moses C. Anyim a, Daniel C. Oshi a,*, Joseph N. Chukwu a, Emmanuel N. Aguwa b,

Inyang N. Johnson c, Charles Nwafor a, Anthony O. Meka a, Chidubem Ogbudebe a,
Nelson O. Madichie a, Ngozi Ekeke a, Samuel B. Olanisebe d
a
German Leprosy & TB Relief Association (GLRA), 35 Hill View, Independence Layout, Enugu, Nigeria
b
University of Nigeria, Enugu Campus, Nigeria
c
SamLans Laboratory, Ibadan, Nigeria
d
Department of Veterinary and Microbiology Parasitology, University of Ibadan, Ibadan, Nigeria

A R T I C L E I N F O A B S T R A C T

Article history: Objective: To assess the quality of week 8 sputum smear AFB microscopy performed by
Received 10 June 2014 peripheral TB laboratories in Nigeria.
Accepted 17 June 2014 Method: A cross-sectional review was performed of all week 8 tuberculosis sputum smear
Available online 12 July 2014 slides reported for the first quarter of 2009 by peripheral laboratories in five States of Nige-
ria. Each slide was reviewed by two independent external slide readers as external quality
Keywords: check and also crosschecked with fluorescent microscopy.
Laboratory Results: In Akwa Ibom, Anambra, Enugu, Kogi and Ogun States, a total of 415, 315, 231, 206
Sputum and 428 week 8 slides respectively were studied (a grand total of 1595 slides studied). The
Conversion rate wide range of conversion rates between the different States as reported by peripheral labs
Nigeria (83.8% in Anambra State to 98.1% in Kogi State) was also observed by the external quality
Tuberculosis check (68.4% in Kogi State to 88.0% in Akwa Ibom State). In all the States, the studied spu-
tum conversion rates reported by the peripheral labs were significantly higher than values
obtained from external quality check and fluorescent microscopy (P = 0.000).
Conclusion/recommendation: There is a wide range of sputum conversion rates between
States, but the conversion rate in each State is significantly higher than those of external
quality check possibly indicating many false negative reports by peripheral labs. It is rec-
ommended that training and re-training of laboratory persons be continued. Internal and
external quality checks should also continue to be practiced in the national TB program.
! 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights
reserved.

Introduction significantly since the onset of HIV and indeed TB is one of the
major killers in patients with HIV. Nigeria is one of the coun-
Tuberculosis (TB) is one of the major causes of morbidity and tries with the highest TB and HIV burden and was ranked
mortality in developing countries. Its incidence has increased fourth among the 22 countries with the highest burden of

* Corresponding author. Tel.: +234 8064243475.

E-mail address: dannyoshi@yahoo.com (D.C. Oshi).
http://dx.doi.org/10.1016/j.ijmyco.2014.06.005
2212-5531/! 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.
 International Journal of Mycobacteriology
TB. Each year in Nigeria, 220,000 positive cases of pulmonary
tuberculosis (PTB) cases occur, of which approximately 50%
are smear-positive [1]. To combat this Nigeria adopted the
WHO recommended TB Control Strategy. One of the com- error (5%) [9]. When the above formula is used, a sample size of
ponents of this strategy is diagnosis through a quality assured
network of TB sputum microscopy services.
Sputum smear microscopy is an important diagnostic pro-
cedure for PTB in the National Tuberculosis and Leprosy Con-
trol Program (NTBLCP) in Nigeria [2]. Suspects diagnosed as
smear positive are started on anti-TB drugs as soon as possi-
ble. A patient is normally treated for 8 months (32 weeks)
according to the national guideline using the four fixed dose
combination (4FDC) treatment regimen [3,4]. A patient started
on treatment, at the end of 8 weeks, submits a sputum sam-
ple for assessment of treatment [5].
Most patients convert to a negative smear result, but a rea-
sonable number of patients do not convert to a negative
smear result at the end of 8 weeks [6,7]. A report of the spu-
tum conversion rate at 8 weeks in the NTBLCP in 2007 showed
a very wide range of 5193%. Many questions come to mind:
can inefficient TB microscopy be responsible? What is the
actual sputum conversion rate at the end of 8 weeks in Nige-
ria? There have been various reports on sputum conversion
rates done in some countries of the world, but as far as this
research is concerned, there has not been any research to
establish reliable conversion rates in Nigeria. The dangers of
reporting false negative/positive results in TB control cannot
be overemphasized.
The aim of this study was to evaluate previous week 8
smear results of new TB patients reported by peripheral labs
in various regions and States in Nigeria.
Materials and methods
Study area
The study site covers all the Directly Observed Treatment
Short course (DOTS) microscopy centers in five selected
States covering the North-central, South-east, South-west
and South-south geopolitical zones in Nigeria.
Study population
The study was conducted in five States sampled from the four
geopolitical zones mentioned above. Kogi State was selected
from the North-Central zone while Enugu and Anambra
States were selected in the south-east of Nigeria. Ogun State
was selected from the south-west zone. Akwa Ibom State
was selected from the South-south zone.
Study design
The study design is a descriptive cross-sectional study involv-
ing a review of smear microscopy of sputum samples at week 8.
Sample size estimation
The number of patients to be sampled was guided by the upper
limit required to give a 95% level of confidence at an expected
3 ( 2 0 1 4 ) 1 7 8 1 8 3 179
conversion of about 85.0% [8] using the precise prevalence for-
mula: Sample size (N) = Z2P (100 ! P)/D2 where Z is a constant
given as 1.96, P is expected conversion 85%, and D is acceptable
195.9 is obtained. To make up for errors in data completion, all
complete slides (i.e., two week 8 slides per patient) for the first
quarter of 2009 from these selected States were studied.
Sampling method used for the States
The thirty-six States in Nigeria were stratified to correspond
to low, medium and high ranges of sputum conversion rates
at week 8 based on the statistical report for 2007, published
by the NTBLCP. The 2009 sputum conversion rate report has
since been released and it showed a similar wide range of
sputum conversion rates for new smear positive (Category
1) PTB cases. However, the 2009 report is incomplete for some
States and so cannot be used for the study. Two States were
selected from each stratum using a simple random method.
The States are: for low range (51% to <75%) Kogi; for medium
range value (75% to <85%) Ogun and Enugu; for high range
values (8593%) Anambra and Akwa Ibom.
Only the slides that were complete (i.e., two week 8 slides
per patient) in all the five States for the first quarter of 2009
were selected. All slides used were stored for not more than
four months; they are slides stored for the existing quarterly
external quality assurance (EQA) of the NTBLCP in the year.
Ethical consideration and inclusion/exclusion criteria
Approval was obtained from the Ethics Committee of the Uni-
versity of Nigeria Teaching Hospital and any other relevant
regulatory agency. Inclusion criteria were: the patient must
have been AFB smear positive before the start of treatment
and the patient must be a new case, i.e., Category 1. Those
excluded were: all transferred cases; all Retreatment and Cat-
egory 2 cases; months 3, 5 and 7 cases; smear negative at
diagnosis and cases with incomplete slides.
Data collection
All week 8 slides were picked across TB smear microscopy
labs in each State, for rechecking using fluorescent micros-
copy and the light microscopy. The slides were picked by
the State Laboratory focal persons (SLFP) in the five States.
The SLFP sent the slides to Irrua Specialist Teaching Hospital
by courier. The re-checking was done by two independent
readers (to minimize error due to variation inherent in multi-
ple readers). A third reader read any discordant slides arising
from the two independent readers. Slides selected from all
the States were re-checked at Irrua Specialist Teaching Hospi-
tal. All slides picked from this study area for re-checking were
de-oiled using xylene and dried. The slides were re-stained
using ZiehlNeelsen as well as Fluorescence method.
Quality check
For quality check the slides are read by two independent read-
ers; any discordance is re-checked by a third person, who
serves as a gold standard.
180 International Journal of Mycobacteriology 3 ( 2 0 1 4 ) 1 7 8 1 8 3

Quality assurance
The laboratory and the staff are part of the NTLCP; the hospi-
tal laboratory is part of the network of peripheral labs that
perform TB smear microscopy in Nigeria and they are part
of an ongoing external quality assurance.
Recording of results
ings is recorded as the result, e.g. where one slide is + and the
second slide is ++ then the recorded result is ++.
Data analysis
The data generated were entered and analyzed in a Statistical
Package for Social Sciences (SPSS). Chi-square test was used
to compare the previous readings to present readings.

Semi-quantitative results were recorded according to the Results

National TB Programme (NTP) guidelines. If one or both slides
collected from a patient is positive, the result is recorded as In Akwa Ibom (South-south region) 415 second-month slides
positive (as in NTLCP). Also, the higher value of the two read- were studied. The sputum conversion rate recorded by

Table 1 Comparison between week 8 AFB peripheral microscopy results and EQC results for Akwa Ibom State.
Akwa Ibom State week 8 AFB microscopy results comparison
Peripheral lab External quality check External quality
(microscopy) (EQC) result check (EQC) result
(light microscopy) (fluorescent microscopy)

AFB test result Number (%) Number (%) Number (%)

Negative 394 (94.9) 365 (88.0) 322 (77.6)
Positive 21 (5.1) 50 (12.0) 93 (22.4)
Total 415 (100.0) 415 (100.0) 415 (100.0)
Comments: conversion rate 94.9% 88.0% 77.6%

v2 and P value.
1. Between peripheral lab and EQC light microscopy results = 12.95 (P value = 0.000) Significant.
2. Between peripheral lab and EQC fluorescent microscopy results = 52.71 (P value = 0.000) Significant.

Table 2 Comparison between week 8 AFB peripheral microscopy results and EQC results for Anambra State.
Anambra State week 8 AFB microscopy results comparison
Peripheral lab External quality check External quality
(microscopy) (EQC) result check (EQC) result
(light microscopy) (fluorescent microscopy)

AFB test result Number (%) Number (%) Number (%)

Negative 264 (83.8) 238 (75.6) 238 (75.6)
Positive 51 (16.2) 77 (24.4) 77 (24.4)
Total 315 (100.0) 315 (100.0) 315 (100.0)
Comments: conversion rate 83.8% 75.6% 75.6%

v2 and P value.
1. Between peripheral lab and EQC light microscopy results = 6.63 (P value = 0.01) Significant.
2. Between peripheral lab and EQC fluorescent microscopy results = 6.63 (P value = 0.01) Significant.

Table 3 Comparison between week 8 AFB peripheral microscopy results and EQC results for Enugu State.
Enugu State week 8 AFB microscopy results comparison
Peripheral lab External quality check External quality check
(microscopy) (EQC) result (EQC) result (fluorescent microscopy)
(light microscopy)

AFB test result Number (%) Number (%) Number (%)

Negative 214 (92.6) 186 (80.5) 186 (80.5)
Positive 17 (7.4) 45 (19.5) 45 (19.5)
Total 231 (100.0) 231 (100.0) 231 (100.0)
Comments: conversion rate 92.6% 80.5% 80.5%

v2 and P value.
1. Between peripheral lab and EQC light microscopy results = 14.61 (P value = 0.000) Significant.
2. Between peripheral lab and EQC fluorescent microscopy results = 14.61 (P value = 0.000) Significant.
 International Journal of Mycobacteriology 3 ( 2 0 1 4 ) 1 7 8 1 8 3 181

peripheral laboratories was 94.9%. This was significantly
higher than the conversion rate of 88.0% obtained by external
quality check (EQC) light microscopy (v2 = 12.95; P
value = 0.000) and 77.6% by fluorescent microscopy
(v2 = 52.71; P value = 0.000). Similar high conversion rates were
also obtained in peripheral laboratories in Anambra and
Enugu States (both in South-east region). In Anambra State
315 s-month slides were studied. A peripheral laboratory
result recorded 83.8% as against 75.6% recorded by EQC light
microscopy and fluorescent microscopy (v2 = 6.63; P
value = 0.01). In Enugu 231 slides from peripheral laboratories
had a conversion rate of 92.6%, while EQC microscopy labora-
tories and fluorescent microscopy had 80.5% (v2 = 14.61; P
value = 0.000) (Tables 13).
In Kogi State (North-central region) 206 second-month
slides reported a conversion rate of 98.1% by peripheral labo-
ratory. This was significantly higher than 68.4% and 60.7%
conversion rates recorded by EQC (v2 = 64.78; P value = 0.000)
and fluorescent microscopy (v2 = 84.32; P value = 0.000)
respectively. In Ogun State (South-south region) 428 slides
collected showed conversion rates of 90.2% by peripheral lab-
oratories. This again was significantly higher than the conver-
sion rate of 76.2% obtained by EQC and fluorescent
microscopy (v2 = 37.45; P value = 0.000) (Tables 46).

Table 4 Comparison between week 8 AFB peripheral microscopy results and EQC results for Kogi State.
Kogi State week 8 AFB microscopy results comparison
Peripheral lab External quality check External quality
(microscopy) (EQC) result check (EQC) result
(light microscopy) (fluorescent microscopy)

AFB test result Number (%) Number (%) Number (%)

Negative 202 (98.1) 141 (68.4) 99 (60.7)
Positive 4 (0.9) 65 (31.6) 64 (39.3)
Total 206 (100.0) 206 (100.0) 163 (100.0)
Comments: conversion rate 98.1% 68.4% 60.7%

v2 and P value.
1. Between peripheral lab and EQC light microscopy results = 64.78 (P value = 0.000) Significant.
2. Between peripheral lab and EQC fluorescent microscopy results = 84.32 (P value = 0.000) Significant.

Table 5 Comparison between Week 8 AFB peripheral microscopy results and EQC results for Ogun State.
Week 8 AFB microscopy results comparison
Peripheral lab External quality check External quality check (EQC)
(microscopy) (EQC) result (light microscopy) result (fluorescent microscopy)

AFB test result Number (%) Number (%) Number (%)

Negative 386 (90.2) 326 (76.2) 326 (76.2)
Positive 42 (0.8) 113 (23.8) 113 (23.8)
Total 428 (100.0) 428 (100.0) 428 (100.0)
Comments: conversion rate 90.2% 76.2% 76.2%

v2 and P value.
1. Between peripheral lab and EQC light microscopy results = 37.45 (P value = 0.000) Significant.
2. Between peripheral lab and EQC fluorescent microscopy results = 37.45 (P value = 0.000) Significant.

Table 6 Comparison between week 8 AFB microscopy results and EQC results for the 5 States.
Week 8 AFB microscopy results comparison for the 5 States
Peripheral lab External quality check External quality
(microscopy) (EQC) result check (EQC) result
(light microscopy) (fluorescent microscopy)

AFB test result Number (%) Number (%) Number (%)

Negative 1460 (91.5) 1256 (70.9) 1171 (75.5)
Positive 135 (8.5) 339 (21.3) 381 (24.5)
Total 1595 (100.0) 1595 (100.0) 1552 (100.0)
Comments: conversion rate 91.5% 70.9% 75.5%

v2 and P value.
1. Between peripheral lab and EQC light microscopy results = 103.12 (P value = 0.000) Significant.
2. Between peripheral lab and EQC fluorescent microscopy results = 148.46 (P value = 0.000) Significant.
Range (conversion rate):
Peripheral laboratory = 83.8% (Anambra State) to 98.1% (Kogi State).
EQC (light microscopy) = 68.4% (Kogi State) to 88.0% (Akwa Ibom State).
182 International Journal of Mycobacteriology
Discussion
The quality of sputum smear results is a critical element in
the management and control of TB (DOTS strategies). This
is of major concern especially in the developing countries that
still largely depend on sputum smear microscopy for diagno-
sis. A false positive smear result at 8 weeks will be interpreted
as non-conversion and will lead to extending the intensive
phase treatment for an extra month while a false negative
result would mean wrongly converting a patient to the con-
tinuation phase treatment when he/she should have been
given an extended 4 weeks of intensive phase drugs. This
false negative result is particularly worrisome because it
could lead to drug resistant strains of TB (MDR-TB or XDR-TB).
Results in the present study reveal two particularly inter-
esting issues. First, there is a wide range of sputum conver-
sions reported by peripheral labs in various regions of the
country: in Anambra State (South-east region) 83.8% was
reported; while in Kogi State (North-central region) 98.1% con-
version rate was reported. Secondly, in all the regions studied,
the peripheral labs report significantly higher rates of conver-
sion at the end of the 8 weeks of intensive treatment for TB
when compared with results from both external quality
checks and fluorescent microscopy.
In the first issue, one wonders if such is truly the case, and,
if yes, why is there such a wide range of conversions at
8 weeks in these regions within the same country? Inciden-
tally, the wide range of conversion rates recorded by periphe-
ral labs is also reported by the external quality checks and
fluorescent microscopy. Conversion rates have been docu-
mented by some studies to be determined by some factors
like age of patient, pre-treatment bacillary load, rate of
default, presence or absence of resistant strains, etc. A Came-
roonian study observed that the factors of age 40 years and
above and a bacillary load of 3+ on pre-treatment sputum
were significantly associated with non-conversion of sputum
smears at the end of two months of treatment [10]. Another
study established that under field conditions even with DOTs,
new smear positive patients with a heavy bacillary load
showed statistically significant poor sputum conversion rates
at two and three months and higher failure rates as compared
with patients with a lesser bacillary load [11]. Similarly, in
Rwanda, the smear conversion rate was 82%, and this varied
significantly from facility to facility depending on location of
the facility, i.e., rural or urban [12]. Two separate studies in
India also reported two widely different conversion rates at
the end of 8 weeks of treatment: one reported 58.0% [13] while
another reported 84.0% [14].
In the second issue, there were significantly higher conver-
sion rates from all the reports from peripheral labs in all the
regions studied compared with external quality checks and
fluorescent microscopy. Interestingly also, a 2007 report on
some States showed a widely different conversion rate com-
pared with that of a 2009 report, e.g., Kogi State conversion
rate for 2007 was 58% while in 2009 it was 98.1%. Could such
a remarkable improvement in conversion rates within the
same State mean that there is an equally remarkable
improvement in TB case management or false reporting by
3 ( 2 0 1 4 ) 1 7 8 1 8 3
the TB lab persons who may not be diligent enough to exam-
ine the slides properly?
Diligence in studying the TB slides greatly affects the
interpretation of results. Indeed, the sensitivity of sputum
smear microscopy has been reported to vary (range, 20
80%), often depending on the diligence with which speci-
mens are collected, smears are made, and stained smears
are examined [15]. Hence, those lab staff who lack diligence
in slide examination hastily conclude that the slide is neg-
ative when they are actually positive, thus leading to a
higher conversion rate.
The very high conversion rates obtained from peripheral
labs are suspicious because they are significantly different
from external quality checks and fluorescent microscopy
results done on the same slides and also do not agree with
most findings from other parts of the world, especially in
countries with high HIV/TB co-infection like Nigeria. A
study in Oman obtained a sputum smear conversion rate
of 78.6% [16]. In another study done in India the conversion
rate was 58%, 61% and 62% in patients with PTB alone, PTB
plus type 2 diabetes and PTB with HIV infection, respec-
tively [13].
Attempts have been made to improve the quality of TB
microscopy. One method used to improve the quality of TB
microscopy was by integrating malaria microscopy quality
assessment into the existing TB microscopy QA system.
When such a program was implemented, it resulted in an
increase in the specificity of both TB and malaria microscopy
results. At the final assessment, 100% specificity was achieved
for TB microscopy results [1]. However, this needs to be fur-
ther tested in various other settings.
Conclusion and recommendation
A wide range of sputum TB microscopy conversion rates were
observed after 8 weeks of treatment from region to region as
reported by sputum smear microscopy and confirmed by
External Quality Check and fluorescent microscopy. However,
the conversion rates reported by the peripheral labs in all
regions of the country are higher than what was observed
from the quality control checks and fluorescent microscopy
indicating that some of the peripheral results may be false
negative for AFB. It is therefore recommended that training
and re-training of lab persons be carried out. Internal and
external quality checks should also routinely be conducted.
Study limitations
Niger State was originally selected in the research work to
make it a total of six States, but unfortunately could not pro-
duce slides at the end of the intensive phase because such
slides were not stored or produced for rechecking. The
National TB control program requests a quarterly report from
the peripheral laboratories; hence, it is not certain if some of
the collected slides are second or third month AFB results.
This, however, did not affect the study since the external
quality checks were done on the same slide irrespective of
month of collection.
 International Journal of Mycobacteriology
Funding
This research was funded by the German Leprosy and TB
Relief Association, Nigeria.
Conflict of interest
The authors declare no conflict of interest.
Author contributions
M.C.A., J.N.C., D.C.O., E.N.A. and I.N.J. conceived the study. The
study protocol was designed by M.C.A., D.C.O., I.N.J., J.N.C.,
C.C.N. and E.N.A. M.C.A., I.N.J. and S.B.O. carried out the labo-
ratory study and oversaw data collection. E.N.A. performed
data entry and carried out the data analysis. D.C.O., M.C.A.,
E.N.A., A.O.M., N.O.M., C.O. and N.E. did the data interpreta-
tion and conceptualization of the manuscript outline.
E.N.A., M.C.A. and D.C.O. drafted the manuscript, while
D.C.O., M.C.A., E.N.A., J.N.C., C.C.N., A.O.M., N.O.M., C.O. and
N.E. critically revised the manuscript for intellectual content.
All authors read and approved the final manuscript. D.C.O. is
guarantor of the paper.
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